This application entitled "Regulating IRS-proteins by Ser/Thr Phosphorylation" is the amened competing renewal ofDK38712-16. IRS1 is a prototype docking protein that links the activated insulin receptor tyrosine kinase to down-stream signaling pathways. Tyrosine phosphorylation of IRS 1, and its homolog IRS2, activates the PI3K and ras/MAPK cascades, which promote cellular growth, survival and metabolism. Nutrient excess, acute and chronic inflammation, or proinflammatory cytokines regulate IRS-protein signaling through multisite Ser/Thr-phosphorylation. During the previous funding period, we revealed the inhibitory role of Ser307 phosphorylation in rodent IRS1 (Ser312 in human IRS1), and showed the utility of polyclonal phosphospecific antibodies to study these processes in murine and human cells and tissues. Phosphorylation of Ser307 is mediated, at least in part, by the association of the activated NH2-terminal JUN kinase (JNK) with IRS1, linking insulin signaling to the inhibitory effects of metabolic stress and proinflammatory cytokines. IRS1and IRS2 each contain at least 40 Ser/Thr-phosphorylation sites (probably more), but how they interact to regulate insulin action in various tissues under acute and chronic metabolic stress is important to resolve. Here, we propose 5 Specific Aims to reveal how Ser/Thr-phosphorylation of Irs1 and Irs2 modulates insulin sensitivity and contirbutes to life threatening metabolic diseases that can progress to diabetes. 1. Prepare and validate a comprehensive mAb library against observed S/T-phosphorylation sites in Irs1 and Irs2 (apS/TmAbIrs1 and apS/TmAbIrs2 libraries). Support is requested in year 1 to generate the library against Irs1 (apS/TmAbIrs1);the apS/TmAbIrs2 library already underway and supported by other sources. 2. Utilize the pS/TmAblrs1 and pS/TmAbIrs2 libraries to decode the S/T-phosphorylation that occurs on Irs1 and Irs2 during metabolic stress. 3. Investigate the regulatory function of Ser307 in Irs1 using mice in which the Irs1 alleles are replaced with mutant A307Irs1 alleles. 4. Determine whether Ser302 in Irsl plays a positive or negative regulatory role upon Irsl signaling in murine tissues. 5. Establish the role of the Jnk-directed "kinase interaction motif (KIM) in Irsl. This proposal is important because dysregulated insulin signaling contributes to life threatening metabolic disease that progresses to type 2 diabetes when pancreatic b-cells fail to secrete sufficient insulin quickly enough to compensate for insulin resistance. Ser/Thr-phosphorylation of Irs 1 and Irs2 is common cause of insulin resistance. The production and validation of the apS/TmAbIrs1 and apS/TmAbIrs2 libraries to detect and quantify the phosphorylation sites provides a rational experimental platform to decoding the complex regulatory mechanism that controls the insulin response throughout life.